Derivatized pyridinoline reagent

ABSTRACT

A pyridinoline composition in which pyridinoline is derivatized specifically at its aliphatic hydroxyl group by a selected chemical group is disclosed. In various embodiments, the composition may be used as a standard for HPLC or immunoassay of pyridinoline, a pyridinoline immunogen for producing anti-pyridinoline antibodies, and a solid-phase reagent for use in an immunoassay kit. Also disclosed are methods for making and using the composition.

This is a division of application Ser. No. 08/234,068 filed Apr. 28,1994, now U.S. Pat. No. 5,502,197, which is a division of Ser. No.07/954,790, filed Sep. 30, 1992, now U.S. Pat. No. 5,350,855.

FIELD OF THE INVENTION

The present invention relates to assays for measuring pyridinolinecompounds in a sample, and in particular, to acylated pyridinolinecompounds and reagents for use in such assays.

REFERENCES

Black, D., et al., Anal. Biochem. 169: 197-203 (1988).

Black, D., et al., Annals of Rheumatic Diseases 48: 641-644 (1989).

Brown, J. P., et al., Lancet 1091-1093 (1984).

Campbell, A., Monoclonal Antibody and Immunosensor Technology, Elsevier(1991).

Cook, J., et al., Ann. Clin. Biochem. 12: 219 (1975).

Daniloff, Y., et al., Connect. Tissue. Res. 27: 187 (1992).

Eyre, D. R., J. Clin. Endocrinol. Metab. 74: 470A-470C (1992).

Eyre, D. R., et al., Anal. Biochem. 137: 380-388 (1984).

Eyre, D. R., et al., FEBS 2: 337-341 (1987).

Fujimoto, D., et al., Biochem. and Biophys. Res. Commun. 76: 1124-1129(1977).

Fujimoto, D., et al., J. Biochem. 83: 863-867 (1978).

Fujimoto, D., et al., J. Biochem. 94: 1133-1136 (1983).

Gosling, J., Clin. Chem. 36(8): 1408- (1990).

Gunja-Smith, Z., et al., Biochem. J. 197: 759-762 (1981).

Harlow, E., et al., Antibodies: A Laboratory Manual, Cold Spring HarborLab (1988).

Henkel, W., et al., Eur. J. Biochem. 165: 427-436 (1987).

Hojo, et al., Chem. Lett. 133: 437- (1977).

Macek, J., et al., Z. Rheumatol. 46: 237-240 (1987).

Ogawa, T., et al., Biochem. Biophys. Res. Commun. 107: 1251-1257 (1982).

Previero, A., et al., Biochem. Biophys. Acta 263: 7-13 (1972).

Roberts, J. D., and Caserio, M. C., Basic Principles of OrganicChemistry, 2nd Ed., W. A. Benjamin, Inc., Menlo Park, Calif., pp.308-309 (1977).

Robins, S. P., Biochem J. 207: 617-620 (1982a).

Robins, S. P., in Collagen in Health and Disease (Weiss, J. B., et al.,eds.) pp. 160-178, Churchill Livingstone, Edinburgh (1982b).

Robins, S. P., Biochem. J. 215: 167-173 (1983).

Robins, S. P., et al., Ann. Rheum. Dis. 45: 969-973 (1986).

Robins, S. P., et al., Biochim. Biophys. Acta. 914: 233-239 (1987).

Segel, I., Biochemical Calculations, John Wiley and Sons, (1976).

Seibel, et al., J. Rheumatol 16: 964-970 (1989).

Wong, S. S., Chemistry of Protein Conjugation and Cross Linking, CRCPress, Boca Raton, Fla. (1991).

BACKGROUND OF THE INVENTION

There are a variety of conditions in humans which are characterized by ahigh level of bone resorption and by an abnormal balance between boneformation and bone resorption. Among the more common of these areosteoporosis, Paget's disease, and conditions related to the progress ofbenign and malignant tumors of the bone and metastatic cancers whichhave been transferred to bone cells from, for example, prostate orbreast initial tumors. Other conditions which are associated withchanges in collagen metabolism include osteomalacial diseases, rickets,abnormal growth in children, renal osteodystrophy, and a drug-inducedosteopenia. Irregularities in bone metabolism are often side effects ofthyroid treatments and thyroid conditions per se, such as primaryhypothyroidism and thyrotoxicosis as well as Cushing's disease.

It has been recognized that disorders of bone resorption or otherconditions characterized by an abnormal balance between bone formationand bone resorption can be detected by altered levels of pyridiniumcrosslinks in urine (Robins, 1982b; Macek; Black). The crosslinks takethe form of compounds containing a central 3-hydroxy pyridinium ring inwhich the ring nitrogen is derived from the epsilon amino group oflysine or hydroxylysine (Fujimoto, 1978; Robins, 1982a; Gunja-Smith;Ogawa; Eyren).

The pyridinium crosslink compounds found in urine can be grouped intofour general classes: (1) free, native crosslinks having a molecularweight of about 400 daltons (Fujimoto), (2) glycosylated crosslinks andcrosslink peptide forms having a molecular weight of between about 550and 1,000 daltons (Robins, 1983), (3) crosslink peptide forms having amolecular weight between 1,000 and 3,500 daltons (Robins, 1983, 1984,1987; Henkel; Eyre), and (4) crosslink peptide forms having a molecularweight greater than 3,500 daltons. In normal adults, these forms accountfor about 38% (1), 40% (2), 15% (3), and 7% (4) of total urinarycrosslinks. About 80% of the free crosslinks in normal adults ispyridinoline (or Pyd), derived from a hydroxylysine residue, and about20%, deoxypyridinoline, or Dpd, derived from a lysine residue, and thisratio of Pyd/Dpd applies roughly to the other three classes ofcrosslinks in urine. The higher molecular weight crosslinks can beconverted to free crosslinks by acid hydrolysis (Fujimoto, 1978).

Methods for measuring pyridinium crosslinks in urine have been proposed.One of these methods involves the measurement of total hydrolysed Pyd,i.e., Pyd produced by extensive hydrolysis of urinary crosslinks, byquantitating the hydrolysed Pyd peak separated by HPLC (Fujimoto, 1983).The relationship between total hydrolysed Pyd to age was determined bythese workers as a ratio to total hydrolysed Pyd/creatinine, wherecreatinine level is used to normalize crosslink levels to urineconcentration and skeletal mass. It was found that this ratio is high inthe urine of children, and relatively constant throughout adulthood,increasing slightly in old age. The authors speculate that this maycorrespond to the loss of bone mass observed in old age.

Studies on the elevated levels of total crosslinks in hydrolyzed urineof patients with rheumatoid arthritis has been suggested as a method todiagnose this disease (Black). The levels of total hydrolyzed crosslinksfor patients with rheumatoid arthritis (expressed as a ratio of totalcrosslinks measured by HPLC to creatinine) were elevated by a factor of5 as compared to controls. However, only total hydrolysed Pyd, but nottotal hydrolysed Dpd, showed a measurable increase.

In a more extensive study using hydrolyzed urines, Seibel et al. showedsignificant increases in the excretion of bone-specific total hydrolysedPyd crosslinks relative to controls in both rheumatoid andosteoarthritis, but the most marked increases for total hydrolysed Pydwere in patients with rheumatoid arthritis (Seibel).

More recently, the applicants have shown that a variety of bone collagendisorders, including osteoporosis, Paget's disease, osteoarthritis,hyperparathyroidism, and rheumatoid arthritis, can be detected on thebasis of characteristic levels of urinary native Pyd or native Dpd.Levels of native Pyd or Dpd were measured by HPLC separation andquantitation of treated urine samples. The use of native crosslinks fordetection of these bone disorders is advantageous in that theseveral-hour hydrolysis step needed to convert pyridinoline crosslinksto hydrolysed Pyd is avoided.

Assay methods, such as those just noted, which involve HPLC quantitationof crosslinks from hydrolysed samples, or crosslink subfractions fromnon-hydrolysed samples, require accurate calibration of the HPLC peakheights, in order to accurately quantitate each of the peaks. Ideally,an internal standard for use in an HPLC assay of urinary pyridinolineshould (a) be recoverable in substantially the same yield as Pyd and Dpdduring chromatographic fraction of a urine sample, (b) have similarspectroscopic (e.g. UV-visible absorbance and fluorescence) properties,and (c) be characterized by a retention time close to but distinct fromthe retention times of Pyd and Dpd in chromatographic analysis (e.g.,reversed phase C-18 HPLC).

Immunoassays have also been proposed for measuring urinary crosslinks.U.S. Pat. No. 4,973,666 discloses an assay for measuring bone resorptionby detection in urine of specific pyridinium crosslinks, characterizedby specific peptide extensions, associated with bone collagen. Twospecific entities having peptide extensions presumed to be associatedwith bone collagen are described. These are obtained from the urine ofpatients suffering from Paget's disease, a disease known to involve highrates of bone formation and destruction. The assay relies onimmunospecific binding of crosslink compounds containing the specificpeptide fragment or extension with an antibody prepared against thecrosslink peptide. It is not clear whether and how the concentration ofcrosslink peptide being assayed relates to total urinary crosslinks.

Robins has described a technique for measuring pyridinoline in urine bythe use of an antibody specific to hydrolysed Pyd (Robins, 1986). Themethod has the limitation that the antibody was found to be specific forthe hydrolyzed form of Pyd, requiring that the urine sample being testedfirst be treated under hydrolytic conditions. The hydrolytic treatmentincreases the time and expense of the assay, and precludes measurementson other native pyridinium crosslinks. More recently, the applicantshave disclosed an enzyme immunoassay for detection of native Pyd inurine samples, for use in detecting a variety of bone collagendisorders.

The typical enzyme immunoassay format for detecting Pyd involves asolid-phase reagent containing surface-bound Pyd and a soluble anti-Pydantibody, where sample Pyd competes with the surface-bound Pyd forbinding to the soluble antibody. The extent of binding of antibody tothe solid support thus provides a measure of Pyd concentration in thesample. In this format, it is desirable that the surface-bound Pydresemble free Pyd in its antigenic characteristics, i.e., that itsconjugation to the solid surface does not appreciably perturb itsreaction affinity for the soluble antibody.

It is also desirable that the anti-Pyd antibody employed in theassay--whether a polyclonal or monoclonal reagent--has a high bindingaffinity for pyridinoline relative to more complex pyridinolinecrosslinks, i.e., crosslinks containing additional glycosylation orpeptide moieties, particularly when assaying non-hydrolysed samples. Inpreparing such an antibody reagent, the immunogen (which is typically apyridinoline moiety conjugated to a carrier protein) should berelatively unobstructed at its pyridine ring and attached amino acidpositions. At the same time, the immunogen should be structurallyhomogeneous, to reduce the range of antibodies which are produced inresponse to the immunogen, particularly when the immunogen is used forpreparing a polyclonal antibody reagent.

SUMMARY OF THE INVENTION

The present invention includes a pyridinoline composition in whichpyridinoline is derivatized specifically at its aliphatic hydroxyl groupby a selected chemical group. For use as an internal standard, thederivatized pyridinoline is characterized by a retention time close, tobut distinct from, the retention times of pyridinoline anddeoxypyridinoline, as determined by reversed phase C-18 liquidchromatography. A preferred chemical group for this embodiment is anacetyl group.

In another embodiment, where the chemical group serves to anchor thepyridinoline to a solid support, the composition is useful in a solidsupport for an enzyme immunoassay.

In yet another embodiment, where the chemical group serves to anchor thepyridinoline to a carrier protein, the composition is useful for raisingantibodies against pyridinium crosslink species. Thus, the inventionincludes a pyridinoline-protein conjugate having the followingstructure: ##STR1## wherein "Prot" is a carrier polypeptide, L is alinker, and the pyridinoline-protein conjugate is effective to serve asan immunogen for raising an antibody specific for the pyridinolinemoiety in the conjugate.

In another aspect, the invention includes a method of derivatizing thealiphatic hydroxyl group of pyridinoline. In the method, anhydroussample of pyridinoline is reacted with an acylating agent in anhydroustrifluoroacetic acid for a time sufficient to selectively derivatize thealiphatic hydroxyl group of the pyridinoline.

In one embodiment, the method is useful for preparing an acylatedpyridinoline for use in quantitating pyridinoline in a urine sample.

In another embodiment, the method is useful for preparing a pyridinolineimmunogen or a pyridinoline solid-surface reagent for immunoassayapplications as above.

Also forming part of the invention is a method of quantitatingpyridinoline crosslinks present in a urine sample. The method includesadding to the sample a known quantity of an O-acetylated pyridinoline,resolving the pyridinoline crosslinks and acetylated pyridinoline in thesample by reversed phase C-18 HPLC, and quantitating the separatedcrosslinks by use of the separated acetylated pyridinoline to calibratethe recovery of the crosslinks.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reaction schemes for acylating the aliphatic hydroxyl groupof pyridinoline using acetyl chloride and propionyl chloride.

FIG. 2 shows relative elution times in reversed phase C18 HPLC of Ac-Pyd(peak 2) and Pr-Pyd (peak 3) relative to pyridinoline (peak 1).

FIGS. 3A-3B show a numbering scheme 3A) and a high field ¹ H nuclearmagnetic resonance spectrum (3B) of Ac-Pyd in deuterium oxide solvent.

FIG. 4 shows reversed phase C18 HPLC chromatograms of a mixture of Pydand Dpd (upper trace) and of purified Ac-Pyd (lower trace) prepared by amethod of the present invention. Peak identification: 1, Pyd; 2, Dpd; 3,Ac-Pyd.

FIGS. 5A-5C show reversed phase C18 HPLC chromatograms of afractionated, unhydrolyzed human urine sample (5A), the urine samplewith added Ac-Pyd (5B), and the urine sample with added Pr-Pyd (5C).Peak identification: 1, glycosylated Pyd; 2, Pyd; 3, Dpd; 4, Ac-Pyd; 5,Pr-Pyd.

FIGS. 6A-6C show reversed phase C18 HPLC chromatograms of crosslinksextracted from hydrolyzed urine samples by cellulose chromatography. InFIG. 6A, the extraction procedure included a wash step with THF. In FIG.6B, the extraction procedure included a wash step with DMF (same urinesample). FIG. 6C shows baseline resolution of Pyd, Dpd, and Ac-Pyd froman extracted hydrolyzed urine sample.

FIG. 7 shows a correlation between expected and observed levels ofpyridinoline in a urine sample based on added Ac-Pyd as a standard in anHPLC assay.

FIG. 8 illustrates the stability of Ac-Pyd in various mixtures of aceticacid, water, and added acetic anhydride. A: acetic acid; B: acetic acidcontaining 0.1% acetic anhydride; C: 90% acetic acid in water,additionally containing 0.1% acetic anhydride; and D: 90% acetic acid inwater (no acetic anhydride added).

FIGS. 9A and 9B illustrate reaction schemes for preparing derivatizedcarrier proteins. FIG. 9A shows a reaction of a carrier protein withreagents to produce a thiolated carrier protein. FIG. 9B shows areaction of a carrier with a bifunctional reagent to produce a carrierprotein containing a maleimide group.

FIGS. 10A-10F illustrate reaction schemes for preparingpyridinoline-carrier protein conjugates.

FIGS. 11 illustrates a solid support surface of the present invention.

FIG. 12 shows standard curves for Pyd and Ac-Pyd in an immunoassay usinganti-Pyd antibodies. The assay involved a competitive format in whichPyd had been immobilized on a solid support via the amino groups of Pyd,and in which standard Pyd or Ac-Pyd competed with the immobilized Pydfor soluble anti-Pyd antibodies.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

"Pyd", "Pyd-OH", "pyridinoline", and "free pyridinoline" refer to thecrosslink compound shown at I below, where the ring nitrogen is derivedfrom the θ amino group of a hydroxylysyl residue.

"Dpd", "deoxypyridinoline", and "free deoxypyridinoline" refer to thecrosslink compound shown at II below, where the ring nitrogen is derivedfrom the ε amino group of a lysyl residue. ##STR2##

"Free crosslinks" refers to either compounds I or II or a mixture of thetwo.

"Glycosylated pyridinoline" and "glyco-Pyd" refer to glycosylated formsof compound I.

"Pyd-peptides" and "pyridinoline-peptides" refer to peptide-derivatizedforms of compound I, in which one or more of the three amino acidresidues in the compound is linked via a peptide linkage to additionalamino acid residues.

"Pyd crosslinks" refers to the pyridinium crosslinks in urine whichcontain compound I either in free or derivatized form. Pyd crosslinksinclude Pyd, glyco-Pyd and Pyd-peptides. Similarly, "Dpd crosslinks"refers to the pyridinium crosslinks in urine which contain compound IIeither in free or derivatized form.

"Total H-Pyd" refers to total hydrolysed Pyd produced by hydrolyzing Pydcrosslinks to Pyd. Similarly, "total H-Dpd" refers to total hydrolysedDpd produced by hydrolyzing Dpd crosslinks to Dpd.

"Hydrolysed-Pyd" and "H-Pyd" refer to Pyd produced by hydrolysing Pydcrosslinks in 6N HCl at 110° C. for ˜16-18 hours, or by isolating Pydfrom sheep bone by the method of Black et al. (1988). Similarly,"Hydrolysed-Dpd" or "H-Dpd" refers to Dpd produced by hydrolysing Dpdcrosslinks in 6N HCl at 110° C. for ˜16-18 hours, or by isolating Dpdfrom sheep bone by the method of Black et al. (1988).

"Hydrolyzed crosslinks" refers to H-Pyd and H-Dpd together.

"Un-hydrolyzed urine" refers to urine samples that were not subjected tohydrolysis conditions.

"Native Pyd" and "N-Pyd" refer to Pyd obtained from urine which has notbeen subjected to hydrolytic conditions. Similarly, "Native Dpd" and"N-Dpd" refer to Dpd obtained from urine which has not been subjected tohydrolytic conditions.

"Native crosslinks" refers to N-Pyd and N-Dpd together.

"Pyridinium crosslinks" refers to pyridinium crosslinks which containcompounds I and/or II in free and/or derivatized form.

"Ac-Pyd", "acetylated Pyd", and "O-acetylated Pyd" refer to H-Pyd orN-Pyd that has been acetylated at the aliphatic hydroxyl group, i.e.,the lysyl side chain hydroxyl group of Pyd.

"Pr-Pyd", "propionylated Pyd", and "O-propionylated Pyd" refer to H-Pydor N-Pyd that has been propionylated at the aliphatic hydroxyl group.

"O-acylated Pyd" refers to H-Pyd or N-Pyd that has been acylated at thealiphatic hydroxyl group.

II. Preparation of Acylated Pyridinolines

FIG. 1 shows reaction schemes for preparing exemplary O-acylatedpyridinoline derivatives of the present invention. In general, suchderivatives are prepared by adding a thoroughly dried sample ofpyridinoline (Pyd) to a mixture containing anhydrous trifluoroaceticacid (TFA) and an acylating agent (e.g., acetyl or propionyl chloride).Typically, the ratio of TFA to the acylating agent is about 9:1 (v:v).The reaction vial is then sealed and allowed to react for a timesufficient to selectively acylate the aliphatic hydroxyl group of Pyd.Suitable reaction times vary, but are typically 20-60 minutes induration (see Examples 1 and 2). The reaction is then stopped by theaddition of water, and the solvent is removed by evaporation.

The extent of conversion of Pyd to the corresponding O-acylatedderivative can be assessed by reversed phase C18 HPLC. Specifically, asillustrated in FIG. 2 and detailed in Example 1, acylation of Pyd withacetyl chloride or propionyl chloride produces acylated derivativeshaving retention times close to but distinct from that of Pyd. Thus, thetime course of the reaction can be monitored by following the loss ofPyd and the appearance of the acylated derivatives. In addition,preparative amounts (e.g., milligram quantities) of acylated Pyd can beobtained having greater than 97% purity, as described in Example 2.

The acylation method is characterized by an extent of acylation of Pydstarting material of greater than 80% completion. However, to obtainsuch a high extent of acylation, anhydrous reaction conditions must beemployed. Use of non-anhydrous TFA or of incompletely dried Pyd candiminish or even preclude attainment of the desired product. Inaddition, the reaction should not be allowed to proceed for too long atime (˜several hours) in order to avoid degradatior of the acylatedproduct, and thus, a decreased yield. Thus, an optimal reaction timeshould be determined for a particular set of reaction conditions bymonitoring by HPLC analysis a trial reaction mixture for several hours.Carboxylic acid bromides can be used in place of the acid chlorides,generally leading to faster acylation rates than observed for thecorresponding acid chlorides.

The acylation procedure of the present invention can be used to producea variety of pyridinoline derivatives selectively acylated at thealiphatic hydroxyl group of Pyd. Acylating reagents having variouslinear, branched chain, or cyclic alkyl groups (e.g., n-butanoylchloride and isopropanoyl chloride, or cyclohexanecarbonyl chloride) aswell as reagents having aromatic groups (e.g., benzoyl chloride) can beused to produce Pyd derivatives having different chromatographicretention times than Pyd. In addition, as described in a later section,Pyd can be derivatized with other chemical groups to anchor Pyd to acarrier protein for use as an immunogen, or to anchor Pyd to a solidsupport.

Evidence that the acylation conditions described above afford selectiveacylation of the aliphatic hydroxyl group of Pyd (and not the ringhydroxyl, for example) was obtained by UV-visible and ¹ H-NMRspectroscopies.

It has been shown that aqueous Pyd undergoes a bathochromic shift whenthe pH is changed from acidic (pH˜1) to neutral or alkaline (pH 7.4 or˜13) (Fujimoto et al., 1977). This shift is attributed to conversion ofthe Pyd ring-hydroxyl group (pKa˜4.2) from the protonated form (λmax˜295 nm) to the unprotonated form (λmax˜325 nm), a shift that cannotoccur if the ring hydroxyl group has been acylated.

Accordingly, to determine whether the ring-hydroxyl group had beenacetylated in the acylation procedure of the present invention,UV-visible spectra of acetylated Pyd were recorded under acidic andneutral pH conditions (Example 3). As detailed in Example 3, theUV-visible absorbance behavior of acetylated Pyd closely resembles thatof Pyd itself. Under acidic conditions (pH˜3), the spectrum ofacetylated Pyd showed an absorbance peak at 294 nm. Moreover, when thepH was adjusted to about 7, the peak shifted to 326 nm. These resultsshow that the ring-hydroxyl oxygen in acetylated Pyd is not acetylated.

To obtain further evidence that only the aliphatic hydroxyl group of Pydhad been acylated, acetylated Pyd prepared as in Example 2 wascharacterized by high-field ¹ H NMR spectroscopy (FIGS. 3A and 3B).

                  TABLE 1                                                         ______________________________________                                        Pyd.sup.a                                                                             Ac-Pyd.sup.a                                                                             peak properties.sup.a                                      δ-Shift                                                                         δ-Shift                                                                            (mult., integral, assignment)                              ______________________________________                                        8.10    8.35       (s, 1, ring-2 or 6: ArH)                                   8.05    8.28       (s, 1, ring-2 or 6: ArH)                                   ˜4.5                                                                            ˜4.8 (dd, 1, ring-N: NCH.sub.2).sup.b                           4.17    4.54       (dd, 1, ring-N: NCH.sub.2).sup.c                           3.99    4.36       (t, 1, ring-4: RCH(NH.sub.3.sup.+, CO.sub.2.sup.-)         3.92    5.36       (m, 1, R.sub.2 CHOH/R.sub.2 CHOAc)                         3.79    4.14       (t, 1, ring-5: RCH(NH.sub.3.sup.+, CO.sub.2.sup.-)         3.76    4.04       (t, 1, ring-N: RCH(NH.sub.3.sup.+, CO.sub.2.sup.-)         3.22    3.40       (d, 2, ring-4: CH.sub.2).sup.a                             2.84    3.04       (m, 1, ring-5: Ar--CH.sub.2)                               2.73    2.93       (m, 1, ring-5: Ar--CH.sub.2)                               2.04    2.19       (m, 2, ring-5: CH.sub.2 CH(NH.sub.3.sup.+,                                    CO.sub.2.sup.-)*                                           1.96    2.19       (m, 1, ring-N: CH.sub.2 CH(NH.sub.3.sup.+,                                    CO.sub.2.sup.-)*                                           1.84    2.03       (m, 1, ring-N: CH.sub.2 CH(NH.sub.3.sup.+,                                    CO.sub.2.sup.-)*                                           1.58    1.98       (m, 1, ring-N: γ-CH.sub.2)                           1.47    1.88       (m, 1, ring-N: γ-CH.sub.2)                           --      1.96       (s, 3 expected, CH.sub.3 C(═O)O)*                      ______________________________________                                         .sup.a See structure I at FIG. 1. The peack multiplicities and                integrations for AcPyd were essentially the same as for Pyd.                  .sup.b RingN: NCH.sub.2 proton, obscured by HOD peak.                         .sup.c RingN: NCH.sub.2 proton, not obscured by HOD peak.                     *The indicated integration values for these peaks were measured               individually only with Pyd. Individual integration of these peaks was not     possible for AcPyd due to peak overlaps; however, for AcPyd, integration      of the asteriskmarked peaks together summed to 9.6. The sharp singlet at      1.96 ppm was observed only for AcPyd.                                    

With reference to Table 1, it can be seen that the shift values for manyof the proton groups had not changed significantly after acetylation ofPyd. Notably, however, Ac-Pyd exhibited a new singlet at 1.96 ppm,consistent with the presence of an acetyl methyl group. Although thispeak could not be integrated separately, integration of the singlet andsurrounding peaks (˜2.25 to ˜1.8 ppm) showed a value of 9.6, very closeto the value of 9.0 expected for an acetyl methyl group (3 hydrogens)plus three methylenes (6 hydrogens). In addition, the peak at 3.92 ppmfor the methine hydrogen vicinal to the aliphatic hydroxyl group of Pydwas absent from the spectrum of Ac-Pyd, and instead, a new peak at 5.36ppm had appeared. This large change in δ-shift is expected forconversion of the aliphatic hydroxyl group to an acetyl group (Robertsand Casserio, 1977). These results, together with those obtained aboveby UV-visible spectroscopy, show that Pyd is selectively acylated at itsaliphatic hydroxyl group under the acylation conditions of the presentinvention.

III. Use of an O-Acylated-Pyridinoline as an Internal Standard forCrosslinks on HPLC

In one aspect of the invention, an acylated pyridinoline derivative, asexemplified by O-acetylpyridinoline, is employed as an internal standardfor quantitating pyridinoline crosslinks fractionated by HPLC.

This use of the acetylated derivative is illustrated by an assay methodfor quantitating native or hydrolysed Pyd in a urine or serum sample, asa diagnostic tool for detecting bone collagen disorders. In the assaymethod, a body-fluid sample, preferably a urine sample, is combined witha known amount of the acetylated derivative. The sample material is thenpretreated, e.g., by contacting the sample components with cellulose ornitrocellulose, to remove some of the non-crosslink components in thesample, particularly peptide components. However, some Pyd may be lostduring pretreatment by non-specific binding of Pyd to the solid phasepretreatment material (e.g., cellulose powder). Studies conducted insupport of the present invention have shown that the amount of Pydremoved by the pretreatment step is variable, but does correlate closelywith the amount of derivatized Pyd which is removed in pretreatment (seeExample 7). Thus, the derivatized Pyd serves in part as an internalstandard to correct for losses of Pyd which can occur on pretreatment.

The pretreated material is then analyzed by fractionation by HPLC, asexemplified by reversed phase C18 HPLC, to separate the crosslinkcomponents in the sample. Examples 5 to 9 below describe several HPLCfractionations of hydrolysed and non-hydrolysed urine samples, afteraddition of derivatized-Pyd internal standard and pretreatment bycellulose chromatography.

In the method described in Example 4, Ac-Pyd or Pr-Pyd was added to apre-extracted, unhydrolyzed urine sample and then analyzed by C18 HPLC.As seen in FIGS. 5A-5C (see Example 4 for details) Ac-Pyd (peak 4, FIG.5B) migrated with a retention time distinct from both the retentiontimes of Pyd and Dpd (peaks 2 and 3, respectively), and also from theretention times of the other fluorescent components of the urine sample.Pr-Pyd (peak 5, FIG. 5C) migrated uniquely with respect to Pyd andAc-Pyd, but comigrated with a urine component.

An exemplary method for extracting and analyzing crosslinks in a urinesample is described in Example 5. In the method, the urine sample ismixed with 1 volume of 90% acetic acid containing 200 nM Ac-Pyd, and 4volumes of butanol. The mixture is loaded onto a cellusose column, andthe column is washed with mobile phase (4:1:1 butanol:acetic acid:water)to elute non-crosslink components. The residual butanol is removed bywashing the column with dimethylformamide (DMF). FIG. 6A shows arepresentative HPLC chromatogram of a hydrolysed urine sample containingAc-Pyd added as an internal standard, obtained according to theprocedure just outlined. Both Pyd and Dpd in the sample are cleanlyresolved from one another and from Ac-Pyd. In quantitating either Pyd orDpd, the peak areas of the three peaks can be integrated, and therelationship between peak area and total Pyd can be determined from theAc-Pyd peak. Details are given in Example 5.

FIGS. 6B and 6C are HPLC chromatograms of hydrolysed urine samplescontaining high levels of creatinine (about 25 mM). In the FIG. 6Bchromatogram, the procedure discussed above with respect to FIG. 6A wasused, except the residual butanol is removed by washing the column withtetrahydrofuran (THF). The FIG. 6C chromatogram was obtained as above,by removing residual butanol with DMF. As seen, the cleaner separationis achieved, at high creatinine sample levels, when the residual butanolin the column is removed by DMF washing. Details are given in Example 6.

The accuracy and reliability of the method, for quantitating Pyd by HPLCis demonstrated by the study in Example 7. Here a series of urinesamples containing known amounts of Pyd and a known fixed amount ofAc-Pyd were fractionated by HPLC, as above, including cellulosepretreatment, and the amount of Pyd recovered was determined from thepeak area for Pyd in the chromatogram, corrected for the peak area ofthe Ac-Pyd calibrant. The corrected Pyd values are plotted against theexpected Pyd values (the amount of Pyd originally present in the sampleprior to cellulose pretreatment) in FIG. 7. A least squares analysisgives a slope of 1 with an R value of 0.997.

FIG. 8 shows the stability of the Ac-Pyd compound under differentsolvent conditions. In each of the four plots, Ac-Pyd was held in anHPLC reservoir in the four solvents described in Example 8. At varioustimes up to 5 days, aliquots were analyzed by HPLC and the amount of Pydquantitated by peak area. As seen from FIG. 8, the Ac-Pyd wassubstantially unchanged over a five day period in 90% acetic acid/10%water, but was degraded to various degrees in the other solvents (whichcontained acetic anhydride). The results show the importance of avoidingacetic anhydride in the storage solution.

It can be appreciated from the foregoing that the derivatized Pydprovides a number of advantages as an internal standard for Pyd, in theabove Pyd assay using HPLC. First, the derivative is recoverable insubstantially the same yield as Pyd and Dpd after pretreatment steps andin HPLC chromatographic separation. Secondly, the derivative ischaracterized by a retention time close to, but distinct from, theretention times of urinary Pyd and Dpd crosslinks in chromatographicanalysis (e.g., reversed phase C-18 HPLC). Finally, the derivative hasspectroscopic (e.g. UV-visible absorbance and fluorescence) propertiessimilar to Pyd.

IV.A. Preparation of a Pyridinoline Immunogen

For use in obtaining antibodies selective for Pyd, a pyridinolineimmunogen (hapten) can be prepared by conjugation of a carrier molecule,typically a carrier protein such as keyhole limpet hemocyanin or bovineserum albumin, to the aliphatic hydroxyl group of Pyd. The Pyd may benative or hydrolyzed Pyd, for selectively generating antibodies thatbind native and hydrolyzed Pyd, respectively.

Exemplary methods for coupling the aliphatic hydroxyl group of Pyd to acarrier protein are outlined in FIGS. 9A-9B and 10A-10F. In one generalapproach, the carrier protein and the aliphatic hydroxyl group of Pydare derivatized such that the resultant derivatives can be coupled viareaction of a sulfhydryl group with a maleimide group or an activatedsulfhydryl group. In another general approach, the aliphatic hydroxylgroup of Pyd is converted to a sulfhydryl group which can then becoupled to a carrier protein as above. An excellent compilation ofcoupling reagents and methods can be found in Wong (1991), which isincorporated herein by reference as representative of background art.

FIGS. 9A and 9B illustrate well known methods by which the amino groupsof a carrier protein can be derivatized to contain sulfhydryl groups ormaleimide groups (Wong, 1991, and references therein). In FIG. 9A, thecarrier is reacted with succinimidyl 3-(2-pyridyldithio)propionate(SPDP) to produce acylated adduct V. Treatment of V with dithiothreitol(DTT) yields a thiolated (i.e., sulfhydryl-containing) protein VI,"Prot-SH". In FIG. 9B, the carrier is reacted with N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate VII (SMCC) to produce acarrier VIII that contains maleimide groups.

FIGS. 10A-10D illustrate methods by which the aliphatic hydroxyl groupof Pyd can be derivatized and then coupled to the derivatized carriersof FIGS. 9A and 9B. In FIG. 10A, Pyd is acylated with4-chloroacetylphenylmaleimide (IX) in anhydrous TFA to givemaleimide-containing derivative X. Derivative X is then reacted with athiolated carrier (Prot-SH) to produce immunogen XI.

FIG. 10B shows how other maleimide-containing acylating agents, e.g.,N-succinimidyl maleimidoacetate (Sigma Chemical Co.), N-succinimidyl3-maleimidopropionate (Aldrich Chemical Company or Sigma),N-succinimidyl 4-p-maleimidophenyl)butyrate (Pierce Chemical Co.) andthe like, can be prepared from commercially available compounds. Ingeneral, the N-hydroxylsuccinimide group of XII is removed byhydrolysis, and the resultant carboxylic acid is converted to an acidchloride XIII using thionyl chloride. The acid chloride is then used toproduce a Pyd-containing immunogen as in FIG. 10A.

FIG. 10C shows a reaction scheme in which the aliphatic hydroxyl of Pydis derivatized with an acylating agent XIV,4-nitro-3-(l-chlorocarbonyloxyethyl)phenyl!methyl-3-(2-pyridyldithiopropionicacid) ester, that contains a masked sulfhydryl that is activated towardsthiol exchange. The resultant carbonate XV is then reacted with HS-Prot,(e.g. VI from FIG. 9A) to produce an immunogen having a disulfidelinkage.

Derivatization of Pyd with another masked-sulfhydryl-containing agent isshown in FIG. 10D. Following preparation of acid chloride XVIII fromN-succinimidyl 3-(2-pyridyldithio) propionate XVII, the acid chloride isreacted with Pyd to produce acylated Pyd XIX. The 2-thiopyridine groupis then removed using dithiothreitol (DTT), and the resultant Pydderivative is reacted with a maleimide-containing carrier (SMCC-Prot) toproduce immunogen XX. FIGS. 10C and 10E thus illustrate two was in whichan acylated Pyd containing a masked thiol (e.g., structures XV and XIX)can be coupled to a carrier protein.

FIGS. 10E and 10F illustrate schemes in which the aliphatic hydroxylgroup of Pyd is converted to a sulfhydryl group. In FIG. 10E, Pyd istreated with N,N-dimethylthiourea XXI in HF/pyridine, thereby producingPyd-SH XXII, a sulfhydryl analog of Pyd. This analog can then be coupledto a maleimide-containing carrier (SMCC-Prot) to produce immunogenXXIII.

In FIG. 10F, the aliphatic hydroxyl is tosylated using tosyl chloride inTFA. The resultant tosylated Pyd is then reacted with thioacetate toproduce a thioacetyl-Pyd which is then treated with aqueous sodiummethoxide to remove the acetyl group, thereby producing Pyd-SH forcoupling to a carrier as in FIG. 10E.

The Pyd-protein conjugates prepared by above methods can be used toprepare monoclonal or polyclonal antibodies by conventional methods(e.g., Harlow, 1988).

VI.B. Preparation of Pyd-Coated Solid-Phase Reagent

The Pyd derivatization method forming part of the invention can also beapplied to forming a Pyd-coated solid-phase reagent. The reagent may beused, for example, as an affinity chromatography reagent, forpurification of anti-Pyd antibodies, or in another example, may be usedin a solid-phase immunoassay device. The latter application isillustrated in FIG. 11, which shows a solid-phase reagent 10 whichincludes a solid-phase support 12 having surface attached binding Pydmolecules effective to bind anti-Pyd antibodies. A variety of glass andpolymer resin supports having chemically derivatizable groups, orsurfaces effective in protein adsorption may be used as the solidsupport. In one preferred embodiment, the solid support is coated withprotein, e.g., adsorbed human serum albumin which is derivatized withstreptavidin, indicated at 15 in the figure. Pyd is derivatized withbiotin, according to the derivatization methods described above.Briefly, biotin is derivatized to provide maleimide or sulfhydrylgroups, and the modified biotin is reacted with a thiolate or maleimidegroup containing derivative, prepared as above. The biotin-Pyd conjugateis then attached to the solid support through high-affinitystreptavidin-biotin binding, indicated by dashed lines in the figure.

Alternatively, Pyd can be derivatized directly to the support by firstderivatizing a solid support, such as support 12, to contain maleimideor sulfhydryl groups. The support is reacted with Pyd functionalizedwith a thiolate or maleimide group, according to the method of theinvention, to couple the Pyd to the support through an O-linked bond.

As explained above, the acylating agent used in forming thefunctionalized Pyd may be a protected sulfhydryl group, which is thendeprotected, after coupling to Pyd, to produce a thiolated pyridinoline.The thiolated pyridinoline is then reacted with a maleimide groupimmobilized on the solid support. The O-derivatized Pyd on the solidsupport has accessible pyridinium and peptide moieties, and a uniformstructural attachment to the support.

VI.C. Calibration of Immunoassay

The solid-phase immunoassay for detection of Pyd, described above,utilizes a solid-phase reagent having an O-linked Pyd, and an antibodypreferably prepared using the Pyd immunogen of the invention. In atypical assay format, a Pyd-containing sample, which may be hydrolysed,is mixed with an anti-Pyd antibody in the presence of the solid support.The sample Pyd competes with the solid-phase Pyd for binding to thesolid support, to attach the antibody to the support in inverseproportion to the amount of Pyd in the sample.

It is necessary, in carrying out the assay, to establish a standardcurve of Pyd concentration vs. signal measured on the solid support. Inthis aspect of the invention, the standard curve is generated by anacylated pyridinoline of the invention, e.g., Ac-Pyd. Here calibrationsamples containing increasing known concentrations of the acylated Pydare reacted in the assay method, and the signal recorded for thesolid-phase bound material is plotted against the known concentrationsof calibrant. The standard curve so generated is then used fordetermining Pyd concentrations in samples, according to the measuredsolid-phase signal. This approach is detailed in Example 9.

FIG. 12 shows calibration curves for Pyd and Ac-Pyd constructed asindicated above. The curves show the relationship between a given Ac-Pydamount, as measured in the immunoassay, and the same amount(concentration) of free Pyd. The two, in other words, indicate how astandard curve generated for Ac-Pyd can be corrected for determining Pydin an assay. It is noted that a sample, such as a urine sample, may bepretreated, for example by passage through a nitrocellulose membrane,prior to its reaction with the solid-phase reagent. In this case, it isrecommended that the Ac-Pyd calibrations are taken through the samepretreatment procedures.

The following examples illustrate, but in no way are intended to limitthe scope of the invention.

EXAMPLES Materials and Methods

All chemicals and solvents were of analytical or HPLC grade and wereobtained either from the sources listed below, or from other sourcesthat provided reagents of comparably high purity. Anhydroustrifluoroacetic acid (TFA, in 1 mL sealed ampules) and acetonitrile werefrom J. T. Baker; acetyl chloride was from Aldrich (Milwaukee, Wis.);dimethylformamide (silylation grade, sealed under N₂) and heptafluorobutyric acid (HFBA) in 1 mL sealed ampules was from Pierce(Rockford, Ill.). Acetic acid (glacial) was from Fisher Scientific.Distilled, deionized water was used for HPLC, and all HPLC solvents werefiltered through 0.22 micron filters (Millipore type GV, Bedford,Mass.). Microgranular and fibrous cellulose powders (CC31 and CF1,respectively) were obtained from Whatman, Inc.

Preparation of Pyd

Hydrolyzed Pyd was isolated from hydrolyzed powdered bovine or sheepbone as described by Black et al. (1988).

Native Pyd was isolated from un-hydrolyzed urine as follows. Human urinewas filtered through a 3000 kD cut-off filter (Filton Co.) applying 40psi of back pressure. The filtrate was then lyophilized andreconstituted to 1/20 of the original volume with 0.2M acetic acid.

Concentrated urine was then applied onto a Sephadex G-10 2.6×95 cmcolumn equilibrated with 0.2M acetic acid. Elution from the columnmaterial was analyzed for free Pyd and Dpd as described above (HPLCprotocol C). The free crosslink containing fractions were pooledtogether, adjusted to pH 2.0 and applied onto 1×18 cm cation exchangecolumn (Locarte Co., UK) and equilibrated with 0.1M sodium citrate pH4.2.

Glyco-Pyd, Pyd and Dpd were coeluted thereafter from the ion exchangecolumn with 0.1M sodium citrate pH 4.2. Collected fractions wereanalyzed for the presence of crosslinks by HPLC analysis as above.Fractions containing crosslinks Glyco-Pyd, Pyd, and Dpd were pooledtogether and applied onto 10×2.5 cm reversed phase C18 column (Waters)which was subsequently developed with 2-20% gradient of acetonitrilecontaining 0.1% HFBA. Separated species (glyco-Pyd, Pyd and Dpd) werecollected and concentrated by lyophilization. The dry residues werereconstituted in 0.2M acetic acid and stored at 4° C. Purity of isolatedPyd was measured by gravimetric and elemental analysis, and was found tobe greater than 97%.

High Performance Liquid Chromatography (HPLC)

A. Analytical HPLC

Analytical HPLC was performed using one of three protocols (A, B, andC). Samples were typically diluted with 1-3% HFBA prior being loaded onthe HPLC column.

In protocol A, for analysis of purified Pyd, Dpd, and products ofacylation reactions, the HPLC system included a Hewlett Packard Series1050 injector and pump system linked to a Shimadzu RF 551 fluorescencemonitor, set at 295 nm excitation and 395 nm emission, a Beckman(Fullerton, Calif.) System Gold analog interface system, a Microsorb C18"Short-One" reversed phase column (100×4.6 mm, Rainin Instrument Co.,Inc., Woburn, Mass.), and a 20×2 mm guard column containing C18 packingbetween two 0.5 μm frits. The solvent system consisted of 12%acetonitrile/0.1% HFBA in water at a flow rate of 1.00 mL/min. Thecolumn and tubing leading to the fluorimeter were maintained at aconstant temperature (30° C.) via containment in a temperature controlchamber to minimize changes in fluorescence yield due to fluctuations inambient temperature (Black et al., 1988).

In protocol B, for analysis of hydrolyzed urine samples, the HPLC systemconsisted of a Hewlett Packard 1050 HPLC equipped with pumps,fluorescence detector, and accompanying software. The column, identicalin type to that in protocol A, and tubing were maintained at 30° C. asabove. The elution gradient was as follows: isocratic elution at 3% Bfor two minutes, linear gradient from 3% to 16% B over 1 minute, lineargradient from 16% to 21% B for 8 minutes (separation gradient), lineargradient from 21% back to 3% B over 2 minutes, and equilibration at 3% Bfor 7 minutes; flow rate=1 ml/min; A=0.3% HFBA in water, B=0.3% HFBA in75% acetonitrile. For routine processing of urine samples, the HPLCsystem was linked directly to an automated liquid handling roboticstation (ASPEC), described in Example 5.

In protocol C, for analysis of hydrolyzed or un-hydrolyzed urinesamples, the gradient HPLC system included two Gilson 302 pumps, aShimadzu RF-530 fluorescence monitor (Ex. 295 nm, Em. 400 nm), an AppleIIe controller, and a Microsorb C18 Short-One column plus guard columnas in protocol A. An in-line stainless steel coil (0.4 mm i.d.; 100 μl)immersed in a constant temperature bath (10° C.) was includedimmediately before the fluorimeter to avoid the variations influorescence yield noted in protocol A. A two-stage gradient was used:linear gradient from 17% to 20% B over first 7 minutes, linear gradientfrom 20% to 25% over next 5 minutes, maintain 25% B for 2 minutes, raisesharply to and maintain at 70% B for 6 minutes to remove residualcontaminants, and equilibrate at 17% B for several minutes to return toinitial conditions; flow rate=1 ml/min; A=10 mM HFBA in water, B=10 mMHFBA in 75% acetonitrile.

B. Preparative HPLC

Preparative HPLC (Protocol D) was performed using a Beckman System Goldinterface equipped with a Programmable Solvent Module 126, and a Waters(Milford, Mass.) Nova-Pak HR C18 reversed phase column (100×25 mm). Anelution gradient of 2%-40% acetonitrile in aqueous 0.1% HFBA at 10mL/min over 60 min was used. Fractions (5 mL) were collected every 0.5min using a Bio-Rad (Richmond, Calif.) Model 2100 fraction collector.Use of this method is described in Example 2.

EXAMPLE 1 Small-Scale Preparation of O-Acetylated and O-PropionylatedPyd

O-acetylated (Ac-Pyd) and O-propionylated (Pr-Pyd) forms of Pyd wereprepared on a small scale from the corresponding acid chlorides. Dry Pyd(e.g. 2.5 nmol) in a polypropylene micro-reaction vial was dissolved in0.3 ml of a trifluoroacetic acid/acid chloride mixture (9:1 v/v,prepared immediately before use). The vial was capped, mixed, andincubated for 60 minutes at 20° C. The reaction was stopped by mixingwith a drop of water, and the solution was taken to dryness in acentrifugal evaporator. Typical yields were in the range of 47-76%.Alterations in temperature and time of reaction did not appear toimprove the yield, and the presence of moisture or salts were found todiminish the yield.

To compare the elution properties of Ac-Pyd and Pr-Pyd with Pyd,acylation reactions were carried out with acetyl chloride and propionylchloride as in the preceding paragraph, except that the reactions werestopped by addition of water after 15 minutes so that a substantialamount of unreacted Pyd remained. After the reaction mixtures wereevaporated to dryness, the reaction products were dissolved in 1 ml ofwater, and aliquots were diluted in 1% (w/v) HFBA for HPLC analysis(protocol C).

Elution profiles of the reaction products are shown in FIG. 2, wherepeak 1 is Pyd, peak 2 is Ac-Pyd, and peak 3 is Pr-Pyd. As can be seen,both acylated products eluted after Pyd, with Ac-Pyd eluting about 2minutes (upper trace), and Pr-Pyd about 4.5 minutes (lower trace), laterthan Pyd.

EXAMPLE 2 Large Scale Preparation of O-Acetylated Pyd

Acetylation of Pyd was performed by reacting purified Pyd with acetylchloride in anhydrous trifluoroacetic acid, following the acylationmethod of Previero et al. (1972).

To a thoroughly dried sample of Pyd (10 mg, 0.023 mmol) in a glassscrew-cap vial was added a premixed solution of 9.0 mL anhydroustrifluoracetic acid and 1.0 mL acetyl chloride. The reaction vial wascapped with an air-tight screw-cap teflon seal and allowed to react withstirring at room temperature for 20 min (in contrast to the 60 minutesused in Example 1, a shortened reaction time was sufficient here,possibly because of the much higher concentration of Pyd in theseconditions). The reaction was quenched by adding eight drops of waterover 15 min. The solvent was evaporated using a vigorous stream of Argas followed by further evaporation in a centrifugal evaporator to yielda brown resinous solid. The resinous solid was then dissolved in 200 μldry DMF, followed by the addition of 1.8 mL of 2% HFBA (2.0 ml finalvolume). HPLC analysis (protocol A) of an aliquot of the mixturerevealed the elution profile summarized below:

    ______________________________________                                        Elution time             % total                                              (min)           identity integration                                          ______________________________________                                        7.38            unknown  3.6%                                                 7.65            Pyd      10.7%                                                8.50            AcPyd    81.4%                                                10.26           unknown  1.2%                                                 11.75           unknown  2.3%                                                 13.97           unknown  0.7%                                                 ______________________________________                                    

The product mixture was purified preparatively by HPLC (protocol D).Monitoring the effluent at 296 nm (UV-visible absorbance) revealed asingle major peak (fractions ˜40-65), of which fractions 44-62 werefound to contain AcPyd of at least 98% purity, as judged by analyticalHPLC of individual fractions. Fractions 44-62 were pooled, freeze dried,and the resultant white powder was stored at 4° C. Yield: ˜45-65%.

As an alternative method, purification of Ac-Pyd could be carried outusing a Locarte amino acid analyser (160×9 mm column). Separation ofAc-Pyd from unreacted Pyd was performed using 68 mM tri-sodium citrate,pH 4.25, at 55° C. Fractions containing purified Ac-Pyd were pooled andstored at -20° C.

Both this method and the preparative HPLC method described aboveprovided Ac-Pyd of about the same purity (typically>97% pure). Thefluorescence yield of Ac-Pyd was found to be the same as that of Pyd.

EXAMPLE 3 Structural Characterization of O-Acetylated Pyd

To determine what group(s) on Pyd had been acylated in the reactionconditions described in Examples 1 and 2, acetylated Pyd prepared as inExample 2 was characterized by UV-visible and ¹ H-NMR spectroscopies.

To determine whether the ring hydroxyl group had been acetylated,acetylated Pyd produced as in Example 1 was examined by UV-visiblespectroscopy under acidic and neutral pH conditions. Spectra wererecorded in the range of 200-500 nm using an HP 8452A diode arrayspectrophotometer.

Approximately 12 μg of acetylated Pyd were dissolved in 750 μl of 0.01MHCl (final pH˜3). The mixture was placed in a 1 cm pathlength cuvette,and the UV-vis spectrum was recorded. In the wavelength range of 260-360nm, a single peak was observed (λmax=294 nm, 0.44 AU). HPLC analysis ofan aliquot of this solution dissolved in 2% HFBA showed that nohydrolysis of the acetyl group had occurred. The pH of the cuvettesolution was then adjusted to about 7 by addition of 1M NaHCO₃, and thespectrum was recorded again. The peak at 294 nm was now absent, and anew peak at 326 nm was observed (0.38 AU). This result indicated thatthe ring hydroxyl group of Ac-Pyd was not acetylated.

To further characterize the chemical structure of Ac-Pyd, dried samplesof Pyd and Ac-Pyd (˜3 mg each) were submitted for ¹ H NMR analysis tothe National Center for NMR Applications at the Department of Chemistry,Colorado State University, Fort Collins, Col. High field ¹ H NMR spectrawere recorded on Bruker AM-500 (for Pyd, 500 MHz) and AM-600 (forAc-Pyd, 600 MHz) FT spectrometers. A water-suppression sequence was usedin data collection for Pyd only, and proton decoupling was used to helpidentify some of the peaks. The spectra obtained are summarized in Table1, with shift values listed in ppm downfield from an external standard(DSS) in deuterium oxide.

EXAMPLE 4 HPLC of Ac-Pyd and Pr-Pyd in the Presence of FluorescentUrinary Components

To investigate the suitability of Ac-Pyd and Pr-Pyd for use as standardsfor the quantitation of urinary Pyd and Dpd by HPLC, small amounts ofAc-Pyd and Pr-Pyd were added to aliquots of a sample ofpre-fractionated, unhydrolyzed urine, and each mixture was analyzed byHPLC (Protocol C).

The resulting chromatograms for urine sample alone (trace A), urinesample plus Ac-Pyd (trace B), and urine sample plus Pr-Pyd (trace C),are shown in FIG. 5. Note that peak 1 is glycosylated Pyd, peak 2 isPyd, peak 3 is Dpd, peak 4 is Ac-Pyd, and peak 5 is Pr-Pyd. The Figureshows that under these conditions, Ac-Pyd is a more suitable standardthan Pr-Pyd, since the former elutes in a region that is vacant for theunhydrolyzed urine sample alone, whereas the latter coelutes with anunknown peak present in the urine sample.

EXAMPLE 5 Quantitation of Pyd Using an Automated HPLC Procedure WithO-Acetylated Pyd as an Internal Standard

For this study, an ASPEC™ liquid handling robotic station (Gilson,France) was used to facilitate the extraction of hydrolyzed crosslinksfrom urine samples. The ASPEC was equipped with individual 1 mldisposable extraction columns (Analytichem International, Inc., HarborCity, Calif.), each containing 100 mg of CC31 microgranular cellulosepowder bordered in the column by two polyethylene frits (20 μm poresize). For each sample, the ASPEC was able to add solvents to thesample, load the sample onto an extraction column, treat the column witha solvent sequence to elute hydrolyzed crosslinks, and load thecollected crosslinks onto a reversed phase C18 HPLC column for analysis(protocol B or C).

Hydrolyses of urine samples were carried out by mixing 1 ml of a urinesample with 1 ml of 12N HCl and heating the mixture for 18 hours at 110°C. in a tightly sealed vial. Following hydrolysis, the samples wereallowed to settle by gravity or were centrifuged at 13,500×g for 2 min.Aliquots of the supernatants (0.6 ml) were added to 10×75 mm glass tubesin the sample rack of the ASPEC™. The ASPEC was also loaded with anextraction column for each sample tube. The ASPEC dilutor reservoir wasfilled with mobile phase (1-butanol:acetic acid:water, 4:1:1 v/v/v), andthe solvent rack was loaded with separate bottles of Ac-Pyd standard(200 nM Ac-Pyd in 90% acetic acid), butanol, DMF, and 1% HFBA (forsample dilution). The ASPEC was programmed to perform the followingsteps:

1. Condition column with 1 ml of mobile phase.

2. Add to the sample tube 0.6 ml of Ac-Pyd standard and 2.4 mln-butanol, and mix mixture (3.6 ml) by sparging.

3. Load sample mixture onto column.

4. Wash column with 4 ml of mobile phase (8 ml for concentrated urinesamples).

5. Wash column with 1.5 ml of DMF.

6. Dry column with 3 ml air.

7. Elute crosslinks with 0.6 ml of 1% HFBA.

8. Dry column with 1.5 ml air.

The flow rates for the above steps were adjusted to avoid highback-pressure, and were generally in the range of 0.5-1.5 ml/min.

The presence of butanol in the eluted crosslinks caused severe peakbroadening and loss of peak retention in subsequent reversed phase HPLCanalysis. Accordingly, a wash step using DMF (step 5) was included toremove residual butanol from the column prior to elution of thecrosslinks with 1% HFBA. A drying step (step 6) was also included towash the DMF from the column.

Following dilution in 2% HFBA, extracted crosslink samples werechromatographed as in protocol B. For quantitation of Pyd and Dpd in theurine samples, the fluorescence peak integrator was calibrated forinternal standardization using peak areas obtained from a standardmixture analyzed by the HPLC at the beginning of each batch of samples,thereby establishing a response factor correlating peak area with loadedamount of Pyd and Dpd. The standard mixture contained 120 nM Pyd and 40nM Dpd in water. The Pyd and Dpd contents of a urine sample weredetermined automatically based on peak integration using the formula:##EQU1## where RF is the response factor determined from the Pyd/Dpdstandard mixture, Peak Area is the peak area measured for the Dpd or Pydfrom the urine sample, IS Peak is the peak for the Ac-Pyd internalstandard that had been added to the urine sample in step 2 of the ASPECcellulose chromatography procedure, IS weight is the amount of Ac-Pydadded to the sample in step 2, and Sample Dilution is the amount bywhich the extracted sample was diluted prior to loading on the HPLCcolumn. FIG. 4 illustrates the purity, as measured using HPLC protocolC, of the Pyd/Dpd standard mixture used to calibrate detector response(upper trace) and of the Ac-Pyd internal standard used to quantitatecrosslink recovery (lower trace).

A chromatogram of a typical hydrolyzed urine sample extracted andanalyzed as above is shown in FIG. 6A.

EXAMPLE 6

Analysis of Samples Having High Creatinine Levels

The automated HPLC procedure of Example 5 was used to process andanalyze hydrolyzed urine samples having high creatinine concentrations.DMF, THF, ethanol and acetone were each tested as the wash solvent instep 5 of the extraction procedure. All four solvents produced improvedpeak shape and retention in comparison to what was obtained when thewash step was omitted, with DMF and THF providing the best results.

FIGS. 6B and 6C show HPLC chromatograms of a urine sample having a highcreatinine level (25 mM), where THF (FIG. 6B) or DMF (FIG. 6C) was usedin step 5. As can be seen, the resolution of peaks was good despite thehigh concentration of creatinine originally present in the sample.Moreover, the crosslink mixture afforded by DMF was significantlycleaner than the mixture afforded by THF.

EXAMPLE 7

Crosslink Levels Measured With and Without Ac-Pyd Internal Standard

To characterize the effectiveness of Ac-Pyd as an internal standard forquantitating the recovery of the crosslinks, five aliquots of ahydrolyzed urine sample containing a known concentration of Pyd werespiked with additional amounts of Pyd, yielding final Pydconcentrations: 905, 1275, 1650, 1943, and 2141 nM. The spiked sampleswere then extracted by cellulose chromatography and analyzed by reversedphase HPLC, by the general procedure described in Example 5. As acontrol, the spiked samples were also analyzed by HPLC withoutperforming the extraction procedure.

In general, the observed levels (uncorrected) of Pyd from the extractedsamples were found to be about 80-82% of those originally loaded.Correction of these levels using the integrated peak area for Ac-Pyd bythe method outlined in Example 5 afforded corrected Pyd levels that wereclose (˜100%) to those originally present prior to the celluloseextraction step. The good agreement of expected and observed (corrected)values of Pyd is shown in FIG. 7; the slope of the line was 1.07, andthe correlation coefficient was 0.997.

EXAMPLE 8 Stability of Ac-Pyd in a Storage Solution

Since hydrolyzed urine samples are typically mixed with butanol andacetic acid (final ratio butanol:acetic acid:sample≈4:1:1 v/v/v) forcellulose chromatography, different solvent conditions were investigatedto develop an Ac-Pyd standard solution that would be stable as well asconvenient to use. The most convenient approach appeared to be toinclude the Ac-Pyd standard in the acetic acid reagent added to theurine sample. However, monitoring of Ac-Pyd in acetic acid showed thatabout 20% of Ac-Pyd was lost after about 4 days. This loss was notaccompanied by an increase of free Pyd. Since acetic anhydride is aknown trace contaminant in even the purest grades of acetic acid, theinvolvement of acetic anhydride in the loss of Ac-Pyd was tested bypreparing solutions of Ac-Pyd (120 nM) in (A) acetic acid, (B) aceticacid containing 0.1% added acetic anhydride, (C) 90% acetic acid inwater additionally containing 0.1% acetic anhydride (D) 90% acetic acidin water (no acetic anhydride added). To follow the disappearance ofAc-Pyd, aliquots were removed periodically and analyzed by HPLC(protocol C). The results are shown in FIG. 8.

Trace A shows the rate of loss of Ac-Pyd in acetic acid alone. Toinvestigate whether this loss could be the result of trace amounts ofacetic anhydride in the sample, acetic anhydride was added to a finalconcentration of 0.1%. As seen from trace B, the added acetic anhydridemarkedly increased the rate of loss of Ac-Pyd, such that virtually noneremained after 4 days (trace B). The effect of added acetic anhydridecould be substantially reduced by the inclusion of water (10% finalconcentration, trace C). When the solvent was 90% acetic acid in water(no added acetic anhydride), no detectable loss of Ac-Pyd had occurredafter 5 days (trace D), showing that the trace contaminant (presumablyacetic anhydride) in the acetic acid had been effectively inactivated bythe addition of water. Accordingly, the last conditions 90% acetic acidin water) were selected for the internal standard solution.

EXAMPLE 9 Use of O-Acetylated Pyd as a Calibrant in an Immunoassay forPyd

To test Ac-Pyd as a possible calibrant in an immunoassay forquantitating N Pyd, the relative affinities of Pyd and Ac-Pyd for ananti-N-Pyd antibody were measured using a competitive immunoassay formatin which Pyd has been immobilized on a solid support. The assay isavailable in kit form (the Crosslinks™ assay) from Metra Biosystems,Palo Alto, Calif.

Solutions of purified Pyd and Ac-Pyd in 0.1M phosphate buffer, pH 7,were prepared with the following concentrations: 0, 25, 75, 250, 750,3000 nM. A molar extinction coefficient of 5420 AU/cm-M at 326 nm wasused in measuring concentrations of both compounds. Aliquots (10 ul) ofeach solution were pipetted into microtiter plate wells (3 replicates)coated with a Pyd-bovine serum albumin conjugate, followed by theaddition of 150 ul of PBS solution containing a predetermined amount ofrabbit anti-N-Pyd antiserum. The resultant reaction mixtures wereincubated for 18 hours at 4° C., after which the wells were washed withPBS (3×250 ul) to remove unbound antibodies. The antibodies bound to thesolid support were then developed by incubating the wells for 1 hour atroom temperature with 150 ul of a solution containing alkalinephosphatase-labeled goat-anti-rabbit immunoglobulin conjugate solution.After removal of unbound conjugate with PBS, the wells were incubated atroom temperature for 1 hour with 150 ul of p-nitrophenylphosphate (2mg/mL) in 1M diethanolamine containing 1 MM MgCl₂, pH 9.8. After the 1hour period, the extent of reaction of p-nitrophenylphosphate in eachwell was measured by absorbance at 405 nm.

Representative calibration curves for Pyd and Ac-Pyd assay are shown inFIG. 12. Although the anti-Pyd antibody did not bind Ac-Pyd as stronglyas Pyd, the Figure shows how a calibration curve based on Ac-Pyd as astandard can be used to determine the concentration of Pyd from a urinesample. Specifically, with reference to FIG. 12, an observed absorbanceof about 0.55 OD corresponds to about 900 nM Ac-Pyd, and to about 500 nMPyd.

Although the invention has been described with respect to particularembodiments, it will be appreciated that various changes andmodifications can be made without departing from the invention.

It is claimed:
 1. A pyridinoline-protein conjugate having the followingstructure: ##STR3## wherein Prot is a carrier protein, L is a linker,and said pyridinoline-protein conjugate is effective to serve as animmunogen for raising an antibody specific for the pyridinoline moietyin the conjugate.
 2. The conjugate of claim 1, having the followingstructure: ##STR4## wherein Pyd represents the pyridinoline moiety inthe conjugate.
 3. The conjugate of claim 1, having the followingstructure: ##STR5## wherein Pyd represents the pyridinoline moiety inthe conjugate.
 4. The conjugate of claim 1, having the followingstructure: ##STR6## wherein Pyd represents the pyridinoline moiety inthe conjugate.
 5. The conjugate of claim 1, wherein Prot is keyholelimpet hemocyanin or bovine serum albumin.
 6. The conjugate of claim 5,having the following structure: ##STR7## wherein Pyd represents thepyridinoline moiety in the conjugate.
 7. The conjugate of claim 5,having the following structure: ##STR8## wherein Pyd represents thepyridinoline moiety in the conjugate.
 8. The conjugate of claim 5,having the following structure: ##STR9## wherein Pyd represents thepyridinoline moiety in the conjugate.